Application of near-infrared three-heteroligand iridium (III) complex multi-mode phosphorescent probe in mitochondrial multi-marker common detection

Abstract

The invention discloses application of a near-infrared three-heteroligand iridium (III) complex multi-mode phosphorescent probe in mitochondrial multi-marker common detection, and belongs to the technical field of biology of organic photoelectric functional materials. Through coordination self-assembly of three functional complementary ligands and an iridium (III) metal center, the near-infrared three-heteroligand iridium (III) complex multi-mode phosphorescent probe not only endows the probe with significant near-infrared luminescence characteristics, but also realizes triple function integration, and can be simultaneously used for mitochondrial biological imaging, oxygen concentration detection and ClO <-> identification. According to the invention, near-infrared luminescence, targeting and multi-mode detection are integrated in one probe molecule, so that the practicability and convenience of the probe are improved, and a new technical thought and method are provided for the fields of biological imaging and molecular detection. The probe has a wide application prospect in the fields of tumor diagnosis, drug screening, environment monitoring, food safety detection and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of biotechnology of organic optoelectronic functional materials. Specifically, it relates to the application of near-infrared tris-heteroleptic iridium(III) complex-based multimodal phosphorescent probes in the co-detection of multiple mitochondrial markers. Background Art

[0002] As the core organelle for cell energy metabolism and apoptosis regulation, mitochondrial imaging technology plays a key role in revealing the pathological mechanisms of neurodegenerative diseases, tumorigenesis, etc. Especially as the core site of aerobic respiration, the oxygen concentration fluctuation in mitochondria directly affects the homeostasis of cell energy metabolism, and its abnormal changes have been confirmed to be highly correlated with the disease processes of multiple sclerosis, ischemia-reperfusion injury, etc. Many diseases, such as Alzheimer's disease and multiple sclerosis, are related to the abnormal operation of mitochondria. Although existing commercial mitochondrial-targeted dyes (such as Mito-Track Green) have the ability of sub-organelle localization, they are limited by the photophysical properties of organic fluorophores and have technical defects such as lack of oxygen sensitivity and poor photostability, resulting in the inability to achieve dynamic monitoring of oxygen metabolism in mitochondria. In addition, hypochlorite (ClO - ) as a key marker of mitochondrial oxidative stress, its abnormal accumulation is closely related to inflammatory diseases such as atherosclerosis. However, traditional detection methods mostly rely on in vitro colorimetry, with limitations such as low spatio-temporal resolution (>10 μm) and the inability to track the dynamic changes of intracellular reactive oxygen species in real time. Therefore, studying the changes in the oxygen concentration in mitochondria in living cells and simultaneously tracking the dynamic changes of reactive oxygen species, and developing multimodal phosphorescent probes with the function of synchronous detection of multiple parameters, are of great significance for the early diagnosis of diseases and the development of drug-targeted delivery systems in the era of precision medicine.

[0003] Compared with traditional organic fluorescent probes, transition metal iridium(III) complexes exhibit high phosphorescence quantum efficiency, long excited-state lifetime, strong anti-photobleaching properties, etc. due to their significant spin-orbit coupling effect, showing unique advantages in the fields of cell imaging and molecular detection. Especially its long excited-state lifetime (from hundreds of nanoseconds to dozens of microseconds) can effectively eliminate the interference of nanosecond-level background fluorescence through time-gating technology, and at the same time achieve quantitative detection of oxygen concentration through the quenching effect of oxygen molecules on triplet excitons. Currently, the structures of iridium(III) complexes as probes for targeted mitochondrial bioimaging, oxygen concentration detection, or hypochlorite detection are mostly bis-heteroleptic ionic [Ir(C^N)2(N^N)] + X - (X = PF6 - , Cl -Configurations such as...) and the luminescence is mostly in the visible light region of 450 - 650 nm. There are technical bottlenecks such as single-modal detection, limited penetration depth in biological tissues (<200 μm), and significant autofluorescence interference. In contrast, the tris-heteroleptic iridium(III) complex constructed by the coordination self-assembly of three functional complementary ligands with an iridium(III) metal center can not only obtain near-infrared luminescence through a single large conjugated main ligand to achieve deep tissue penetration, but also endow the iridium(III) complex with richer functionality and biocompatibility through the molecular engineering of the second ligand and the auxiliary ligand. However, there is no reported multi-modal probe based on near-infrared tris-heteroleptic iridium(III) complexes in the existing literature, especially the lack of an integrated system with functions of mitochondrial dynamics tracing, oxygen metabolism monitoring, and hypochlorite detection, resulting in technical blind spots in the multi-parameter correlation analysis of the mitochondrial microenvironment, which urgently needs to be broken through. Summary of the Invention

[0004] Aiming at the problems that the existing iridium(III) complex-based probes generally adopt a bis-heteroleptic configuration and the luminescence is limited to the visible light region (450 - 650 nm), and there are technical problems such as single detection dimension (such as mitochondrial localization imaging, oxygen concentration detection, or hypochlorite recognition), shallow tissue penetration, and significant autofluorescence interference, the present invention aims to provide the application of near-infrared tris-heteroleptic iridium(III) complex-based multi-modal phosphorescent probes in the co-detection of multiple mitochondrial markers.

[0005] To achieve the above object, the present invention adopts the following technical solutions: The present invention provides the application of near-infrared tris-heteroleptic iridium(III) complex-based multi-modal phosphorescent probes in the preparation of products for co-detection of multiple mitochondrial markers. The general formula of the near-infrared tris-heteroleptic iridium(III) complex-based multi-modal phosphorescent probes is [Ir(C 1 ^N 1 )(C 2 ^N 2 )(N 3 ^N 4 )] + PF6 - , and the structural formula is as follows: ; Among them, the (C 1 ^N 1 ) ligand is a main ligand with a large conjugated cyclometal for achieving near-infrared luminescence; (C 2 ^N 2 ) ligand is a ligand containing an aldehyde group recognition group for recognizing hypochlorite; (N 3 ^N 4) The ligand is an auxiliary ligand containing a mitochondrial targeting functional group, which is used for targeted mitochondrial localization and improving biocompatibility.

[0006] The product is for simultaneously measuring, screening and detecting multiple mitochondrial markers in the mitochondria of living cells, and for cell imaging through near-infrared luminescence of a near-infrared tris-heteroleptic iridium(III) complex-based multimodal phosphorescent probe.

[0007] The multiple mitochondrial markers include key markers of oxidative stress.

[0008] The key markers of oxidative stress include oxygen concentration and hypochlorite ion concentration.

[0009] Further, the structural formula of the detection probe is .

[0010] Furthermore, the emission wavelength of the near-infrared tris-heteroleptic iridium(III) complex-based multimodal phosphorescent probe is 650 - 900 nm.

[0011] The product is any one of a reagent, a kit, and a chip for simultaneously detecting multiple mitochondrial markers.

[0012] The present invention provides a kit for detecting the presence of multiple mitochondrial markers in a sample or measuring the content of multiple mitochondrial markers in a sample, and the kit includes the above-mentioned near-infrared tris-heteroleptic iridium(III) complex-based multimodal phosphorescent probe.

[0013] The present invention provides a method for detecting the presence of multiple mitochondrial markers in a sample or measuring the content of multiple mitochondrial markers in a sample, including: (1) Contacting and mixing the kit with the sample to be tested for incubation to obtain an incubated mixture; (2) Measuring the phosphorescence signal of the incubated mixture.

[0014] The incubation temperature is 36 - 38 °C, and the incubation time is 20 - 26 h.

[0015] Further, the incubation temperature is 37 °C, 5% CO2, and the incubation time is 24 h.

[0016] Compared with the prior art, the present invention has the following technical effects: The application of the near-infrared tris-heteroleptic iridium(III) complex-based multimodal phosphorescent probe provided by the present invention in the preparation of a product for co-detection of multiple mitochondrial markers. This near-infrared tris-heteroleptic iridium(III) complex-based multimodal phosphorescent probe has unique structural and performance advantages. In terms of optical properties, the probe has a single large conjugated cyclometalated main ligand HC 1 ^N 1, enabling the probe to generate near-infrared emission under visible light excitation. This property not only reduces the damage of the excitation light source to biological samples and improves safety in biological tissues, but also has stronger tissue penetration ability, reduces light scattering and background noise, improves the clarity and depth of imaging, and provides strong support for deep detection in vivo. In terms of the recognition and detection function, by endowing the second ligand with an aldehyde group-preembedded ClO - recognition active site, enabling the probe to specifically recognize key markers of mitochondrial oxidative stress (such as ClO-), providing an effective means for detecting the oxidative stress state of mitochondria in cells; in terms of targeting and biocompatibility, HN 3 ^N 4 auxiliary ligand containing a mitochondrial targeting functional group, which can not only accurately target mitochondria, but also increase the biocompatibility of the complex. Through the coordination self-assembly of three functional complementary ligands with the iridium(III) metal center, this probe realizes the integration of multiple functions. It can not only simultaneously achieve targeted mitochondrial bioimaging, but also detect oxygen concentration, and has potential detection and recognition of ClO - due to the presence of aldehyde functional groups, and is a multi-modal phosphorescent probe with good performance. This near-infrared tris-heteroleptic iridium(III) complex multi-modal phosphorescent probe can simultaneously monitor multiple biological parameters in cells, such as oxygen concentration, hypochlorite level, etc., improving the comprehensiveness and accuracy of detection; in tumor diagnosis and treatment, this probe can be used to detect the oxygen concentration and hypochlorite level in the tumor microenvironment, providing an important basis for evaluating the hypoxia state and oxidative stress degree of tumors, and has good application prospects.

[0017] The kit provided by the present invention realizes the integration of near-infrared luminescence, multi-modal detection and high biocompatibility, and provides a new technical tool for the field of cell imaging and molecular detection.

[0018] The method for detecting the presence of multiple mitochondrial markers in a test sample or measuring the content of multiple mitochondrial markers in a sample, using a near-infrared tris-heteroleptic iridium(III) complex probe, breaks through the limitations of bis-heteroleptic, realizes near-infrared emission, and has multiple functions such as mitochondrial targeted imaging, oxygen concentration detection and hypochlorite recognition. It has deep tissue penetration, little autofluorescence interference, comprehensive and accurate detection, and provides a more efficient and accurate tool for mitochondrial function research, especially having significant advantages in deep tissue imaging and complex biological environment monitoring. Description of the Drawings

[0019] Figure 1 is the 1 1H NMR spectrum of the near-infrared tris-heteroleptic iridium(III) complex multi-modal phosphorescent probe S1 of the present invention; Figure 2X-ray single crystal diffraction pattern of the near-infrared triple-heteroleptic iridium(III) complex-based multimodal phosphorescent probe S1 of the present invention; Figure 3 Normalized UV-Vis absorption and emission spectra of the near-infrared triple-heteroleptic iridium(III) complex-based multimodal phosphorescent probe S1 of the present invention; Figure 4 Emission spectra of the near-infrared triple-heteroleptic iridium(III) complex-based multimodal phosphorescent probe S1 of the present invention under different oxygen concentrations; Figure 5 Co-stained cell imaging diagrams of the near-infrared triple-heteroleptic iridium(III) complex-based multimodal phosphorescent probe S1 of the present invention and commercial mitochondrial dyes. Detailed implementation manners

[0020] To enable those skilled in the art of the present technology to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0021] For those experimental steps or conditions not specified in the embodiments, the operations or conditions of the conventional experimental steps described in the literature in this field can be followed. For the reagents or instruments not specified for the manufacturer, they are all conventional reagent products that can be obtained through commercial purchase.

[0022] In the present invention, unless otherwise specified, all experimental raw materials used are commercially available products well-known to those skilled in the art.

[0023] The MCF-7 cells used in the present invention were purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. The mitochondrial dye Mito-Tracker was purchased from Beyotime Biotechnology Co., Ltd.

[0024] Example 1 This example provides a near-infrared triple-heteroleptic iridium(III) complex-based multimodal phosphorescent probe, and the structural formula of the near-infrared triple-heteroleptic iridium(III) complex-based multimodal phosphorescent probe is as follows: 。

[0025] 1. The specific preparation steps are as follows: Under N2 atmosphere, 1-chloroisoquinoline (4.0 mmol, 654.4 mg), benzo[b]thiophene-2-boronic acid (4.8 mmol, 854.5 mg) and Pd(PPh3)4 (5.0 mol%, 277.3 mg) were weighed and dissolved in a mixed solvent of toluene-ethanol (60 mL, v / v = 2:1); 2M aqueous sodium carbonate solution was added, and the mixture was stirred at 85 °C in an oil bath for 48 hours to obtain a brownish-yellow reaction solution. After cooling to room temperature, the solution was filtered, and most of the solvent was removed by rotary evaporation. The filtrate was extracted with dichloromethane and distilled water multiple times. The organic phase was dried over anhydrous magnesium sulfate and then concentrated again using a rotary evaporator. The crude product was separated and purified by silica gel column chromatography, and the eluent was ethyl acetate : n-hexane = 1 : 9 (v / v), obtaining 762 mg of white solid with a yield of 73%, obtaining HC 1 ^N 1 Ligand Hiqbt.

[0026] HC with a molar ratio of 1.5:1.5:1 was 1 ^N 1 Ligand Hiqbt, HC 2 ^N 2 Ligand Hfppy (purchased from Aladdin Holdings Group Co., Ltd., ≥95%, P160242), iridium(III) chloride trihydrate were added to a mixed solvent of 20 mL of ethylene glycol monoethyl ether and deionized water (the volume ratio of ethylene glycol monoethyl ether to deionized water is 3:1). The reaction was carried out at 110 °C for 24 h under a nitrogen atmosphere. After the reaction was completed, it was cooled to room temperature, saturated sodium chloride solution was added and stirred for 1 hour, the precipitate was collected and dried in a vacuum drying oven at 45 °C for 12 hours to obtain a dichloro-bridged intermediate mixture; The bridged intermediate mixture with a molar ratio of 1:2.5, HN 3 ^N 4 Auxiliary ligand Hbpy (purchased from Aladdin, ≥99%, D108977) was added to a mixed solvent of 15 mL of dichloromethane and methanol (the volume ratio of dichloromethane to methanol is 2:1). The reaction was carried out at 55 °C for 24 h under a nitrogen atmosphere and then cooled to room temperature. 10 equiv of ammonium hexafluorophosphate was added, and the reaction was stirred at 25 °C for 12 h to obtain a near-infrared triple-heteroleptic iridium(III) complex-based multimodal phosphorescent probe S1.

[0027] 2. Performance testing (1) NMR identification The near-infrared triple-heteroleptic iridium(III) complex-based multimodal phosphorescent probe S1 was dissolved in deuterated chloroform (CDCl3), and the 1H NMR spectrum at room temperature was measured as Figure 1 shown below, and the spectral assignments are as follows: 11H NMR (CDCl3, 400 MHz): δ (ppm) 9.67 (s, 1H, -CHO), 9.10 (d, 1H, -Py), 8.72 (q, 2H, -Py), 8.23 (t, 1H,-Py), 8.11 (m, 2H, -Py), 7.99 (t, 1H, -Ph), 7.89 -7.81 (m, 8H, -Ph), 7.56 (t,1H, -Py), 7.48 (t, 1H, -Py), 7.38 (t, 1H, -Ph), 7.31 (d, 3H, -Py), 7.23 (d,1H, -Ph), 6.92 (t, 1H, -Py), 6.84 (s, 1H, -Py), 6.02 (d, 1H, -Py).

[0028] The 1H NMR of the near-infrared tris-heteroleptic iridium(III) complex-based multimodal phosphorescent probe S1 1 contains the proton peaks of the ligands (iqbt)-, (fppy)- and the auxiliary ligand (bpy), and the molar ratio of the number of protons of the three ligands is exactly 1:1:1. The chemical shift of the -CHO characteristic proton peak on the second ligand (fppy)- is δ = 9.67 ppm, which is consistent with the theory. The above results confirm the correctness of the structure of the target complex.

[0029] (2) X-ray single crystal diffraction The purified near-infrared tris-heteroleptic iridium(III) complex-based multimodal phosphorescent probe S1 was dissolved in DMSO solution, and dark red block single crystals of the near-infrared tris-heteroleptic iridium(III) complex-based multimodal phosphorescent probe were obtained by slow evaporation at room temperature. The crystal structure of the complex was obtained by X-ray single crystal diffraction test, and the data was analyzed and refined to convergence by the crystal analysis software Olex 2.15. The single crystal structure diagram was drawn using the diamond 4.0 software, as Figure 2 shown. The results show that the space group of the complex is P-1. The C^N chelating cyclometalated main ligand (iqbt)- and the second ligand (fppy)- and the N^N chelating auxiliary ligand (bpy) are coordinated to a trivalent iridium central ion together to form a distorted six-coordinate octahedral configuration. This result intuitively proves the correctness of the structure of the tris-heteroleptic ionic iridium(III) complex.

[0030] Example 2 Based on Example 1, the photophysical properties and laser confocal bioimaging of the near-infrared tris-heteroleptic iridium(III) complex-based multimodal phosphorescent probe S1 of the present invention were tested to verify its application.

[0031] (1) Near-infrared luminescence performance The near-infrared tris-heteroleptic iridium(III) complex-based multimodal phosphorescent probe S1 was dissolved in deoxygenated dichloromethane solution (1×10 -5 M), and its room-temperature ultraviolet-visible-near-infrared absorption spectrum (UV-Vis-NIR) and photoluminescence spectrum (PL) are as Figure 3 shown.

[0032] The ultraviolet-visible-near-infrared absorption spectrum of the near-infrared tris-heteroleptic iridium(III) complex-based multimodal phosphorescent probe S1 exhibits broadened absorption peaks: strong absorption peaks at 228, 279, 311 nm are attributed to the spin-allowed 1π-π* transitions of the cyclometalated main ligand Hıqbt, the second ligand Hfppy, and the auxiliary ligand bpy; absorption peaks with medium intensity at 440, 483 nm come from the spin-allowed singlet ligand / metal-ligand charge transfer ( 1 LLCT / 1 MLCT) and the spin-forbidden triplet ligand / metal-ligand charge transfer ( 3 LLCT / 3 MLCT) mixed transitions, indicating that the spin-orbit coupling of the central iridium atom results in a large mixing of spin-forbidden 3 MLCT and high-potential spin-allowed 1 MLCT.

[0033] Under the excitation condition of 365 nm, the near-infrared tris-heteroleptic iridium(III) complex-based multimodal phosphorescent probe S1 exhibits typical near-infrared luminescence without fine structure, that is, the maximum emission wavelength from the 0-0 electronic transition is located at 683 nm, and this emission characteristic mainly comes from 3 LC and 3 MLCT (LC: ligand-centered transition, MLCT: metal-ligand charge transfer) mixed-state transitions. And the near-infrared luminescence increases the penetration depth of biological tissues, making it more suitable for bioimaging.

[0034] (2) Emission spectra under different oxygen concentrations In the CH2Cl2 solution (1×10 -5 M) of the near-infrared tris-heteroleptic iridium(III) complex-based multimodal phosphorescent probe S1, the changes in the emission spectrum intensity under hypoxic, normoxic, and hyperoxic conditions were measured respectively, and the results are as Figure 4 shown. With the increase of oxygen concentration, the intensity of the maximum emission peak of the near-infrared tris-heteroleptic iridium(III) complex-based multimodal phosphorescent probe S1 at 683 nm gradually weakens, indicating that this complex can well sense the change of oxygen concentration.

[0035] (3)Laser confocal bioimaging The digested MCF-7 cells were seeded in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 100 mg / mL penicillin, and 100 mg / mL streptomycin, and cultured in a humidified environment with 5% CO2 at 37 °C. The cells were treated with 10 μM near-infrared triple-heteroleptic iridium(III) complex-based multimodal phosphorescent probe S1 for 1 hour, then treated with 200 nM Mito-tracker Green for 30 minutes, washed three times with PBS solution, and imaged as Figure 4 shown.

[0036] Mito-tracker Green was excited by 488 nm blue light, and the green light emission at 505 - 525 nm was collected. The multimodal near-infrared triple-heteroleptic ionic iridium(III) complex probe S1 was excited by 405 nm blue-violet light, and the near-infrared light emission at 650 - 750 nm was collected. By superimposing the emission regions of the two, a high degree of overlap was found, and the Pearson coefficient was calculated to be 0.84. It was demonstrated that the near-infrared triple-heteroleptic iridium(III) complex-based multimodal phosphorescent probe S1 of the present invention can target live cell mitochondria and can be used for live cell mitochondrial labeling.

[0037] In summary, the present invention innovatively designed and successfully constructed a near-infrared triple-heteroleptic iridium(III) complex-based multimodal phosphorescent probe. Through the coordination self-assembly of three functional complementary ligands with the iridium(III) metal center, this type of probe not only exhibits significant near-infrared luminescence characteristics, but also realizes the integration of triple functions: (1) precise mitochondrial targeting; (2) real-time dynamic monitoring of hypoxic, normoxic, and hyperoxic states in cells; (3) specific recognition of hypochlorite. This technical solution effectively fills the technical gap of triple-heteroleptic ionic iridium(III) complexes in the field of near-infrared biological detection and sensing. Its innovative molecular configuration breaks through the functional limitations of traditional single-modal probes and successfully realizes the multi-parameter synchronous detection ability of diagnostic and therapeutic integrated probes. This achievement not only provides a new molecular tool for the multi-dimensional analysis of the live cell microenvironment, but also provides an innovative molecular construction strategy and theoretical basis for the development of the next generation of multi-modal diagnostic and therapeutic integrated probes.

[0038] The above content is only to illustrate the technical idea of the present invention and cannot be used to limit the protection scope of the present invention. Any modification made on the basis of the technical solution according to the technical idea proposed by the present invention falls within the protection scope of the claims of the present invention.

Claims

1. Application of a near-infrared triple-heteroleptic iridium(III) complex-based multimodal phosphorescent probe in the preparation of a product for co-detection of multiple mitochondrial markers, characterized in that, The general formula of the near-infrared triple-heteroleptic iridium(III) complex-based multimodal phosphorescent probe is [Ir(C 1 ^N 1 )(C 2 ^N 2 )(N 3 ^N 4 )] + PF6 - , and the structural formula is as follows: 。 2. The application according to claim 1, wherein The product is for simultaneously measuring, screening, and detecting multiple mitochondrial markers in the mitochondria of living cells, and for cell imaging through near-infrared luminescence of a near-infrared triple-heteroleptic iridium(III) complex-based multimodal phosphorescent probe.

3. The application according to claim 1, wherein The multiple mitochondrial markers include key markers of oxidative stress.

4. The application according to claim 3, wherein The key markers of oxidative stress include oxygen concentration and hypochlorite concentration.

5. The application according to claim 1, characterized in that, The structural formula of the near-infrared triple-heteroleptic iridium(III) complex-based multimodal phosphorescent probe is .

6. The application according to claim 5, wherein The emission wavelength of the near-infrared triple-heteroleptic iridium(III) complex-based multimodal phosphorescent probe is 650 - 900 nm.

7. The application according to claim 1, characterized in that, The product is any one of a reagent, a kit, and a chip for simultaneously detecting multiple mitochondrial markers.

8. A kit for detecting the presence of multiple mitochondrial markers in a sample or measuring the content of multiple mitochondrial markers in a sample, characterized in that, The kit includes the near-infrared triple-heteroleptic iridium(III) complex-based multimodal phosphorescent probe described in claim 1.

9. A method for detecting the presence of multiple mitochondrial markers in a sample or measuring the content of multiple mitochondrial markers in a sample, characterized in that, Comprising: (1) Contacting and mixing and incubating the kit described in claim 8 with a sample to be tested to obtain an incubated mixture; (2) Measuring the phosphorescence signal of the incubated mixture.

10. The method for detecting the presence of multiple mitochondrial markers in a sample or measuring the content of multiple mitochondrial markers in a sample according to claim 9, wherein the incubation temperature is 36 - 38 °C and the incubation time is 20 - 26 h.

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